The overall goal of this procedure is to build a micro drive that can be used for brain recordings in mice while they move freely within an experimental chamber. This is accomplished by first preparing a bundle of four micro electrodes into a tede. The second step is to construct the base of the micro drive and assemble the drive components.
Next, the tetros are attached to the drive and are connected to gold pins. The final step is to gold plate the tip of the tetros to lower their impedance and ensure high signal to noise recordings. Ultimately, chronic micro drive arrays are used to record the neural activity of populations of neurons, as well as single neurons in awake behaving animals.
The main advantage of this technique is that it offers a fairly inexpensive and light wave solution for chronic brain recordings in mice and other small mammals. This technology can address key issues in the field of neuroscience, such as how neurons encode information during the performance of behavioral Tasks. We regularly employ this technique to record from neurons from the dorsal hippocampus and ulu, but have also used it to target deeper structures such as the amygdala.
Visual demonstration of this method is crucial as the construction and the assembly part are difficult to learn, and also because you are dealing with very small and delicate part To begin tetro fabrication. For the hippocampus cut 30 centimeters of insulated 12.5 micrometer diameter core platinum iridium wire, fold the wire twice to form four parallel wires each 7.5 centimeters in length. Next, attach a rubber coated clip near the bottom of the draped wire and attach it to the motorized tetro spinner, making sure that the wire is taut but not too taut or bearing weight is this will cause it to break.
During the spinning process, apply 80 clockwise rotations to wind the tero, followed by 20 counterclockwise rotations in order to release tension. The final number of rotations per length of wire should be eight rotations per micron. Next, set the heat gun to 400 degrees Celsius and use it to fuse the wires together.
Run the heat gun up and down the length of the wires for five seconds, keeping it approximately two centimeters from the wire at all times. To gently melt the VG bond coating on the wires, cut the top of the tero to form four separate wires, and then release the bottom end from the clip. Place the completed tero in a dust-free holding box for storage until the drive has been completed.
First, construct the base for the micro drive by sanding a 20 millimeter square piece of plexiglass acrylic that is five millimeters thick into a shape that will allow the mouse to move freely with the drive after it is implanted on the head. Next, solder two 3.3 by 6.3 millimeter brass guides together perpendicularly. The vertical brass guide will hold the drive screw and electrodes while the horizontal piece will be glued into the acrylic base.
Then begin assembly of the drive itself by passing a filler head breast screw through the top of the guide and into a delrin plastic block. Thread a hex breast not onto the screw until the nut is nearly touching the bottom of the guide. However, do not tighten it fully.
Instead, melt a small amount of solder in order to join the nut and the screw. But be careful not to solder anything to the guide. Rotating the screw should move the delrin block clockwise and counterclockwise vertically along the thread.
Once the drive has been assembled, go back to the acrylic base and cut a three millimeter wide slot where the electrode drive will be. Then pass the horizontal bra guide through the slot and then use Sano ACRL glue to secure the piece to the base. Next, place an electronic interface board on top of the base and to mark the locations of the two screw holes using a 1.5 millimeter tip drill, bit carefully drill holes at the marks for screws that will hold the EIB in place on top of the base.
Then thread two breast screws into the holes. Use micro dissecting scissors to cut four seven millimeter long pieces of polyamide tubing. Line them up next to each other on a piece of folded laboratory tape and apply sano ACRL to join them together.
Allow enough time for the joint tubes to dry fully. Next, carefully dab a small amount of cyanoacrylate on the delrin face. Then place the four joint tubes on the glue.
Again, allow enough time for the glue to set completely before attempting to move the drive. Test that the polyamide tubes are securely attached and that the entire assembly moves smoothly without touching the guide or meeting any resistance. Next, prepare the ground screw and connect the ground wire to the EIB as described in the accompanying text protocol.
Connect tetros and or single electrodes to the EIB by first passing the wires through the polyamide tube, allowing them to extend at least two millimeters past the end of the tube. Then apply a small drop of cyanoacrylate, affixing the wire to the tube and preventing any wire movement. Next, connect the loose ends of the tero or other wires to an EIB channel hole using a gold pin.
Finally, trim off any excess wire with fine scissors shown. Here are examples of other drive configurations that can be constructed to target different brain regions. To begin versa, drive assembly.
Cut the polyamide tubing to 10 millimeters and guide it through the smallest hole on a tetro carrier until it extends 0.5 millimeters past the end. Then epoxy the polyamide tube in place and repeat these steps for the three other tubes and carriers. After the epoxy has fully set.
Guide each polyamide tube through one of the four holes on the Versa drive base. Then push an insect pin through the outer hole. This will hold the tetro carrier in line and serve as a rail for the carrier to travel on.
Repeat this for the three other carriers. Next, take a cap and line it up with the four insect pins so that the cap covers the base and the tero carriers reside within the cap. Thread a one millimeter by five millimeter machine screw through the appropriate hole in the cap and into the tero carrier.
Repeat this for the other three screws. Turn all of the screws clockwise until tetro carriers are at their top position, and the polyamide tubes are visible through the cap opening. Using fine micro dissecting scissors, cut the tubing just below the base so that all four polyamide tubes are of the same length.
Next, use a dissecting microscope to carefully thread the tetros through the polyamide tubes and secure them in place with a small drop of ano aate. Then cut the tetros so that they only extend 2.0 millimeters past the tubes. Before connecting the tero wires to the gold receptacles.
First, connect the ground wires to the cap. Next, guide all the loose tero wires through their respective receptacle holes on the cap and install the cap by lining up the insect pins holes and press fitting to the base. Finally, trim any excess wire that remains protruding from the top of the cap.
First turn the micro drive screws counterclockwise to their lowest position. Then securely mount the micro drive on a clamp that will allow lowering of the electrode tips into the gold plating solution. Fill one delrin tower with CCO gold solution and the other tower with distilled water and lower the electrode tips into the gold solution.
Next, open the Nano Z program and click on DC electroplate. Set the mode to match impedances the plating current to negative 1.0 microamps and the target to 350 kilo ohms at 1004 hertz. Then set it to run five times at five second intervals with a two second pause in between each run.
Once the program is set up, click auto plate, the program will first read the impedance of each channel. Then apply the specified current to that channel, retest the impedance, and apply current as needed until the target impedance or a lower value is reached. If the impedance drops below 100 kilo ohms, reverse the current polarity to positive 1.0 microamps to remove excess gold particles and then repeat the electroplating Typical final impedance values on a bundle of four 12.5 micrometer wires range from 150 to 325 kilo ohms.
Once all channels have been plated to an acceptable impedance level, close the Nano Z program and disconnect the device. Then raise the electrodes out of the plating solution and lower the tips into the distilled water delrin tower to rinse off excess gold particles Representative neural recordings using the micro drive to measure local field potentials from the mouse. Dorsal ulu are shown here.
The four channels shown in part A are an example of a poorly grounded signal that results in saturated values and extremely noisy signals. The four channels shown in part B, however, are a good example of a well-grounded signal with clearly visible network oscillations in the theta range of four to 12 hertz. On the left is an example of a poor Tero recording.
Because the four electrode wires have registered essentially identical spike wave forms, over a thousand individual spike wave forms were overlaid on top of each other for each channel of the teros. This pattern was likely due to the fusing of the wires together during the insulation melting step of tero fabrication, causing the bundle of wires to effectively act as a single recording electrode. On the right is an example of a good tetro recording showing overlaid spike wave forms from a putative unit with different amplitudes across the four tede wires.
This type of spike recording pattern allows for improved unit discrimination during subsequent offline clustering and separation. After constructing this micro dive, it's important to test each of your electrode channel and make sure they are within a proper impedance range before moving to the implantation surgery. After watching this video, you should feel comfortable building teros and micro electrode drives for chronic implantation in the mouse.
Good luck building your drives and have fun with your experiments.
